Research to continue the development of instrumentation and methods for the sequence analysis of peptide antigens presented to the immune system in association with class I and class II molecules of the major histocompatibility complex is proposed. Routine detection and characterization of disease associated antigens present at the attomole level in a mixture of 10,000 self peptides is the expected outcome. Identification of disease associated antigens is an important first step in the development of vaccines or other immune system modulators that are effective against bacterial and viral infections, cancer, autoimmune disorders and tissue transplant rejection. Multistage chromatography in conjunction with our recently developed, automated peak parking technology plus nanoliter/min, high performance liquid chromatography and high performance capillary electrophoresis interfaced to an ion trap mass spectrometer via an electrospray ionization source will be employed to achieve this goal. Specific aims of the proposed research include the following: (1) to identify melanoma specific antigens presented by the class I molecules, HLA A2.1, A1 and A3, and the class II molecule, DR4, (2) to identify minor histocompatibility antigens associated with graft-vs-host disease and tissue transplant rejection, (3) to identify peptides derived from myelin basic protein as well as cross reactive self peptides that are presented by the multiple sclerosis associated class II molecule, DR15Dw2, and recognized by T-cell clones derived from multiple sclerosis patients, (4) to identify peptides presented by the class I molecule, B27, that are unique to individuals diagnosed with ankylosing spondylitis (5) to identify peptides presented by the class I molecule, A2.1, that are unique to individuals diagnosed as having chronic lymphocytic leukemia, (6) to identify and characterize peptides presented by HLA-A2.1, that have been post translationally modified by either phosphorylation or glycosylation, (7) to identify class II peptides presented by the HLA-DR4Dw4 molecule in the presence and absence of the chaperone molecule, HLA-DM.